Effects of edd and pgi Disruptions on Inosine Accumulation in Escherichia coli

Using an inosine-producing mutant of Escherichia coli, the contributions of the central carbon metabolism for overproducing inosine were investigated. Sodium gluconate instead of glucose was tested as a carbon source to increase the supply of ribose-5-phosphate through the oxidative pentose phosphate pathway. The edd (6-phosphogluconate dehydrase gene)-disrupted mutant accumulated 2.5 g/l of inosine from 48 g/l of sodium gluconate, compared with 1.4 g/l of inosine in the edd wild strain. The rpe (ribulose phosphate 3-epimerase gene)-disrupted mutant resulted in low cell growth and low inosine production on glucose and on gluconate. The disruption of pgi (glucose-6-phosphate isomerase gene) was effective for increasing the accumulation of inosine from glucose but resulted in low cell growth. The pgi-disrupted mutant accumulated 3.7 g/l of inosine from 40 g/l of glucose when 8 g/l of yeast extract was added to the medium. Furthermore, to improve effective utilization of adenine, the yicP (adenine deaminase gene)-disrupted mutant was evaluated. It showed higher inosine accumulation, of 3.7 g/l, than that of 2.8 g/l in the yicP wild strain when 4 g/l of yeast extract was added to the medium.     

操纵子前导区的修饰及PRPP合成途径的加强对大肠杆菌积累L-组氨酸的影响

目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。     

Growth-rate recovery of Escherichia coli cultures carrying a multicopy plasmid, by engineering of the pentose-phosphate pathway

Expression of plasmid-encoded genes in bacteria is the most common strategy for the production of specific proteins in biotechnological processes. However, the synthesis of plasmid-encoded proteins and plasmid-DNA replication often places a metabolic load (metabolic burden) into the cell's biochemical capacities that usually reduces the growth rate of the producing culture (Glick BR. Biotechnol Adv 1995;13:247-261). This metabolic burden may be related to a limited capacity of the cell to supply the extra demand of building blocks and energy required to replicate plasmid DNA and express foreign multicopy genes. Some of these required blocks are intermediaries of the pentose phosphate (PP) pathway, e.g., ribose-5-phosphate, erythrose-4-phosphate. Due to the important impact of metabolic burden on biotechnological processes, several groups have worked on developing strategies to overcome this problem, like reduction of plasmid copy number (Seo JH, Bailey JE. Biotechnol Bioeng 1985;27:1668-1674; Jones KL, Kim S, Keasling JD. Metab Eng 2000;3:328-338), chromosomal insertion of the gene which product is desired, or changing the plasmid-coded antibiotic resistance gene (Hong Y, Pasternak JJ, Glick BR. Can J Microbiol 1995;41:624-628). However, few efforts have been attempted to overcome the reduction of growth rate due to protein over-expression, by modifying central metabolic pathways (Chou C-H, Bennett GN, San KY. Biotechnol Bioeng 1994;44:952-960). We constructed a high-copy number plasmid carrying the gene for glucose-6-phosphate dehydrogenase, zwf, under the control of an inducible trc promoter (pTRzwf04 plasmid). By transforming a wild-type strain and inducing with IPTG, it was possible to recover growth-rate from 0.46 h(-1) (uninduced) to 0.64 h(-1) (induced). The same transformation in an Escherichia coli zwf(-), allows a growth-rate recovery from 0.43 h(-1) (uninduced) to 0.62 h(-1) (induced). We also studied this effect as part of a laboratory-scale biotechnology process: production of a recombinant insulin peptide by co-transforming E. coli JM101 strain with pTRzwf07, a low-copy-number plasmid that carries the same inducible construction as pTRzwf04, and with the pTEXP-MMRPI vector that carries a TrpLE-proinsulin hybrid gene. In this system, production of TrpLE-proinsulin strongly reduces growth rate; however, overexpression of zwf gene recovers with a growth rate from 0.1 h(-1) in the TrpLE-proinsulin induced strain, to 0.37 h(-1) when both zwf and TrpLE-proinsulin genes were induced. In this paper, we show that the engineering of the pentose phosphate pathway by modulation of the zwf gene expression level partially overcomes the possible bottleneck for the supply of building blocks and reducing power synthesized through the PP pathway, that are required for plasmid replication and plasmid-encoded protein expression.     

龟裂链霉菌zwf2基因阻断提高土霉素生物合成

葡萄糖-6-磷酸脱氢酶(G6PDH)是链霉菌磷酸戊糖途径中第一个酶("看家"酶),也是形成NADPH的关键酶,由zwf1和zwf2基因编码.以温敏型质粒pKC1139为基础构建了用于阻断龟裂链霉菌zwf2的重组质粒pKC1139-zwf2',通过大肠杆菌GM2929去甲基化pKC1139-zwf2'后电转至原始龟裂链霉菌M4018感受态细胞,筛选得到转化子.转化子进一步通过PCR鉴定和点杂交印迹分析鉴定,证明是zwf2基因阻断的阳性突变子命名为M4018-△zwf2.以原始菌株为对照,突变子摇瓶发酵结果表明:突变子的葡萄糖-6-磷酸脱氢酶酶活是原始菌的50%左右,但土霉素生物合成水平则提高了27%;在细胞生长方面,二者均在第4d进入生长稳定期而开始大量合成土霉素,发酵结束时细胞菌体浓度基本相同,但突变子的单位菌丝体土霉素生物合成能力则提高了31%.因此,zwf2的阻断有利于土霉素的生物合成,而对细胞生长没有明显影响.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

Abstract The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp , encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO₂. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp .